Genetic Control of Early Embryogenesis in the Red Flour Beetle, Tribolium castaneum

SYNOPSIS. The power of genetic analysis possible with the fruitfly, Drosophila melanogaster, has yielded a detailed understandingof pattern formation controlled by homeotic and segmentationgenes in early embryogenesis. We are studying the genetic regulationof embryogenesis in the red flour beetle, Tribolium castaneum.The dynamic process of germ rudiment formation and sequentialsegmentation exhibited by Tribolium provides a context differentthan Drosophila within which to assess the function of homeoticand segmentation gene homologs. Our analyses of the genes inthe HOM-C suggest many similarities in structure and functionwith the well-characterized Drosophila genes. Abdominal resemblesits Drosophila homolog abdominal-A in functioning to establishsegmental identities in the abdomen, such that in each casemutations result in homeotic transformations to PS6. Althoughthe anterior functional boundary of abdominal-A homologs isprecisely conserved, the domain within which Abdominal is importantextends more posterior than that of abdominal-A. The final expression pattern of the segmentation gene engrailedin Tribolium is identical to Drosophila, suggesting that thesehomologs are involved in a conserved developmental process.However, as expected the development of that pattern is different;engrailed stripes anticipate the formation of each new segmentas they appear sequentially in the elongating germ band. Althoughthe grasshopper even-skipped and fushi tarazu homologs are notapparently important in segmentation, the expression patternsof the Tribolium homologs strongly suggest that they have gaineda role in segmentation in the lineage leading to beetles andflies. Nevertheless, differences between Tribolium and Drosophilain the dynamics of even-skipped expression and the fushi tarazumutant phenotype indicate divergence in the regulation and rolesof these genes.     

Characterization of the Tribolium Deformed ortholog and its ability to directly regulate Deformed target genes in the rescue of a Drosophila Deformed null mutant

 We have analyzed the Tribolium castaneum ortholog of the Drosophila homeotic gene Deformed (Dfd) and determined its expression pattern during embryogenesis in this beetle. Tc Deformed (Tc Dfd) is expressed in the blastoderm and the condensing germ rudiment in a region that gives rise to gnathal segments. During germ band extension Tc Dfd is expressed in the mandibular and maxillary segments, their appendages, and the dorsal ridge. Comparison of insect Dfd protein sequences reveals several highly conserved regions. To determine whether common molecular features reflect conserved regulatory functions we used the Gal4 system to express the Tribolium protein in Drosophila embryos. When Tc Dfd is expressed throughout embryonic ectoderm under the control of P69B, the beetle protein autoregulates the endogenous Dfd gene. In addition, the Drosophila proboscipedia gene (a normal target of Dfd) is ectopically activated in the antennal and thoracic segments. We also compared the ability of the beetle and fly proteins to rescue defects in Dfd ^– mutants by expressing each throughout the embryonic during embryogenesis. Both proteins rescued Dfd ^– defects to the same extent in that they each restore the development of mouth hooks and cirri, as well as cause gain-of-function abnormalities of posterior mouth parts. As before, pb was ectopically activated in the antennal segment. This is the first demonstration of the ability of a heterologous homeotic selector protein to directly regulate a target gene independent of an endogenous Drosophila autoregulatory loop. Received: 11 December 1998 / Accepted: 8 March 1999     

The embryonic expression pattern of labial, posterior homeotic complex genes and the teashirt homologue in an apterygote insect

 During embryogenesis of the fruit fly, Drosophila melanogaster, the homeotic genes are required to specify proper cell fates along the anterior-posterior axis of the embryo. We cloned partial cDNAs of homologues of the Drosophila homeotic gene teashirt and five of the homeotic-complex (HOM-C) genes from the thysanuran insect, Thermobia domestica, and assayed their embryonic expression patterns. The HOM-C genes we examined were labial, Antennapedia, Ultrabithorax, abdominal-A and Abdominal-B. As the expression pattern of these HOM-C genes is largely conserved among insects and as Thermobia is a member of a phylogenetically basal order of insects, we were able to infer their ancestral expression patterns in insects. We compare the expression patterns of the Thermobia HOM-C genes with their expression in Drosophila and other insects and discuss the potential roles these genes may have played in insect evolution. Interestingly, the teashirt homologue shows greater variability between Thermobia and Drosophila than any of the HOM-C genes. In particular, teashirt is not expressed strongly in the Thermobia abdomen, unlike Drosophila teashirt. We propose that teashirt expression has expanded posteriorly in Drosophila and contributed to a homogenization of the Drosophila larval thorax and abdomen. Received: 23 July 1998 / Accepted: 1 November 1998     

Identification of the Drosophila and Tribolium receptors for the recently discovered insect RYamide neuropeptides

One year ago, we discovered a new family of insect RYamide neuropeptides, which has the C-terminal consensus sequence FFXXXRYamide, and which is widely occurring in most insects, including the fruitfly Drosophila melanogaster and the red flour beetle Tribolium castaneum (F. Hauser et al., J. Proteome Res. 9 (2010) 5296–5310). Here, we identify a Drosophila G-protein-coupled receptor (GPCR) coded for by gene CG5811 and its Tribolium GPCR ortholog as insect RYamide receptors. The Drosophila RYamide receptor is equally well activated (EC₅₀, 1 × 10⁻⁹ M) by the two Drosophila RYamide neuropeptides: RYamide-1 (PVFFVASRYamide) and RYamide-2 (NEHFFLGSRYamide), both contained in a preprohormone coded for by gene CG40733. The Tribolium receptor shows a somewhat higher affinity to Tribolium RYamide-2 (ADAFFLGPRYamide; EC₅₀, 5 × 10⁻⁹ M) than to Tribolium RYamide-1 (VQNLATFKTMMRYamide; EC₅₀, 7 × 10⁻⁸ M), which might be due to the fact that the last peptide does not completely follow the RYamide consensus sequence rule. There are other neuropeptides in insects that have similar C-terminal sequences (RWamide or RFamide), such as the FMRFamides, sulfakinins, myosuppressins, neuropeptides F, and the various short neuropeptides F. Amazingly, these neuropeptides show no cross-reactivity to the Tribolium RYamide receptor, while the Drosophila RYamide receptor is only very slightly activated by high concentrations (>10⁻⁶ M) of neuropeptide F and short neuropeptide F-1, showing that the two RYamide receptors are quite specific for activation by insect RYamides, and that the sequence FFXXXRYamide is needed for effective insect RYamide receptor activation. Phylogenetic tree analyses and other amino acid sequence comparisons show that the insect RYamide receptors are not closely related to any other known insect or invertebrate/vertebrate receptors, including mammalian neuropeptide Y and insect neuropeptide F and short neuropeptide F receptors. Gene expression data published in Flybase (www.flybase.org) show that the Drosophila CG5811 gene is significantly expressed in the hindgut of adult flies, suggesting a role of insect RYamides in digestion or water reabsorption.     

Negative regulation ofUltrabithorax expression byengrailed is required for proper specification of wing development inDrosophila melanogaster

In both vertebrates and invertebrates, homeotic selector genes confer morphological differences along the antero-posterior axis. However, insect wing development is independent of all homeotic gene functions, reflecting the ground plan of an ancestral pterygote, which bore wings on all segments. Dipteran insects such asDrosophila are characterized by a pair of wings in the mesothoracic segment. In all other segments, wing development is essentially repressed by different homeotic genes, although in the metathorax they are modified into a pair of halteres. This necessitates that during development all homeotic genes are to be maintained in a repressed state in wing imaginal discs. In this report we show that (i) the function of the segment polarity geneengrailed (en) is critical to keep the homeotic selector geneUltrabithorax (Ubx) repressed in wing imaginal discs, (ii) normal levels of En in the posterior compartment of haltere discs, however, are not enough to completely repressUbx, and (iii) the repression ofUbx byen is independent of Hedgehog signalling through which the long-range signalling ofen is mediated during wing development. Finally we provide evidence for a possible mechanism by whichen repressesUbx. On the basis of these results we propose thaten has acquired two independent functions during the evolution of dorsal appendages. In addition to its well-known function of conferring posterior fate and inducing long-range signalling to pattern the developing appendages, it maintains wing fate by keepingUbx repressed.     

Pair-rule and gap gene mutants in the flour beetle Tribolium castaneum

 Early pattern formation in the Drosophila embryo occurs in a syncytial blastoderm where communication between nuclei is unimpeded by cell walls. During the development of other insects, similar gene expression patterns are generated in a cellular environment. In Tribolium, for instance, pair-rule stripes are transiently expressed near the posterior end of the growing germ band. To elucidate how pattern formation in such a situation deviates from that of Drosophila, functional data about the genes involved are essential. In a genetic screen for Tribolium mutants affecting the larval cuticle pattern, we isolated 4 mutants (from a total of 30) which disrupt segmentation in the thorax and abdomen. Two of these mutants display clear pair-rule phenotypes. This demonstrates that not only the expression, but also the function of pair-rule genes in this short-germ insect is in principle similar to Drosophila. The other two mutants appear to identify gap genes. They provide the first evidence for the involvement of gap genes in abdominal segmentation of short-germ embryos. However, significant differences between the phenotypes of these mutants and those of known Drosophila gap mutants exist which indicates that evolutionary changes occurred in either the regulation or action of these genes. Received: 8 May 1998 / Accepted: 17 June 1998     

Terminal versus segmental development in the Drosophila embryo: the role of the homeotic gene fork head

Summary Mutations of the homeotic gene fork head (fkh) of Drosophila transform the non-segmented terminal regions of the embryonic ectoderm into segmental derivatives: Pre-oral head structures and the foregut are replaced by post-oral head structures which are occasionally associated with thoracic structures. Posterior tail structures including the hindgut and the Malpighian tubules are replaced by post-oral head structures associated with anterior tail structures. The fkh gene shows no maternal effect and is required only during embryogenesis. The phenotypes of double mutants indicate that fkh acts independently of other homeotic genes (ANT-C, BX-C, spalt) and caudal. In addition, the fkh domains are not expanded in Polycomb (Pc) group mutant embryos. Ectopic expression of the homeotic selector genes of the ANT-C and BX-C in Pc group mutant embryos causes segmental transformations in terminal regions of the embryo only in the absence of fkh gene activity. Thus, fkh is a region-specific homeotic rather than a selector gene, which promotes terminal as opposed to segmental development. Offprint requests to: Institut für Biologie II (Genetik), Universität Tübingen, Auf der Morgenstelle 28, D-7400 Tübingen, Federal Republic of Germany     

Embryonic expression of the single Tribolium engrailed homolog

We have cloned and sequenced the single Tribolium homolog of the Drosophila engrailed gene. The predicted protein contains a homeobox and several domains conserved among all engrailed genes identified to date. In addition it contains several features specific to the invected homologs of Bombyx and Drosophila, indicating that these features most likely were present in the ancestral gene in the common ancestor of holometabolous insects. We used the cross-reacting monoclonal antibody, 4D9, to follow the expression of the Engrailed protein during segmentation in Tribolium embryos. As in other insects, Engrailed accumulates in the nuclei of cells along the posterior margin of each segment. The first Engrailed stripe appears as the embryonic rudiment condenses. Then as the rudiment elongates into a germ band, Engrailed stripes appear in an anterior to posterior progression, just prior to morphological evidence of the formation of each segment. As in Drosophila (a long germ insect), expression of engrailed in Tribolium (classified as a short germ insect) is preceeded by the expression of several homologous segmentation genes, suggesting that similar genetic regulatory mechanisms are shared by diverse developmental types. © 1994 Wiley-Liss, Inc.     

TGFβ signaling in Tribolium: vertebrate-like components in a beetle

The cytokines of the TGFβ superfamily are highly conserved in evolution and elicit a diverse range of cellular responses in all metazoa. In Drosophila, the signaling pathways of the two TGFβ subfamilies, Activins and Bone Morphogenetic Proteins (BMPs), have been well studied. To address the question of whether the findings from Drosophila are representative of insects in general, we analyzed the components of TGFβ-signaling present in the genome of the beetle Tribolium castaneum. We were able to identify orthologs of the BMPs Decapentaplegic and Glass bottom boat, of the Activins Activinβ and Dawdle, as well as orthologs of the less well-known ligands Myoglianin and Maverick, together with orthologs of all TGFβ receptors and cytoplasmic signal transducers present in Drosophila. This indicates that the diversity of TGFβ signaling components is generally well conserved between Drosophila and Tribolium. However, the genome of the beetle—and of the bee Apis mellifera—lacks an ortholog of the Drosophila BMP Screw but does contain a vertebrate-like BMP10 homolog which is not found in Drosophila. Concerning BMP inhibitors, Tribolium displays an even more vertebrate-like ensemble of components. We found two orthologs of the vertebrate DAN family, Dan and Gremlin, and show embryonic expression of a vertebrate-like BAMBI ortholog, all of which are absent in Drosophila. This suggests that Tribolium might have retained a more ancestral composition of TGFβ signaling components and that TGFβ signaling underwent considerable change in the Drosophila lineage. Tribolium is an excellent model to study the function of these ancestral signaling components in insects. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The <Emphasis Type="Italic">Tribolium spineless</Emphasis> ortholog specifies both larval and adult antennal identity

The morphology of insect antennae varies widely among species, but our understanding of antennal development comes almost solely from studies of one species—the fruit fly, Drosophila melanogaster. Moreover, this knowledge applies mostly to adult structures, since Drosophila lacks external larval appendages. In contrast to Drosophila, the red flour beetle, Tribolium castaneum, has both larval and adult antennae, which are very different from one another in morphology. Thus, Tribolium provides an ideal system to compare modes of antennal development both within and between species. Here, we report that the Tribolium ortholog of spineless (Tc-ss) is required in both the larval and adult antennae. Knockdown of Tc-ss by RNAi during either larval or imaginal development causes transformation of the distal portion of the antennae to legs. Thus, the function of ss is conserved between Drosophila and Tribolium with respect to adult antennal specification and also between Tribolium larval and adult antennal development. The similarity of the Tc-ss RNAi phenotype to that of a classically described Tribolium mutation, antennapedia (ap) (of no relationship to the Drosophila Hox gene of the same name), led us to characterize the original ap mutation and two newly identified ap alleles. Our mapping and phenotypic data suggest that Tc-ss is the best candidate for the ap locus. These results represent a first step in characterizing larval and adult antennal patterning in Tribolium, which should provide important insights into the evolution of insect antennal development.     

Sites of Fgf signalling and perception during embryogenesis of the beetle Tribolium castaneum

The development of multicellular embryos depends on coordinated cell-to-cell signalling events. Among the numerous cell-signalling pathways, fibroblast growth factors (FGFs) are involved in important processes during embryogenesis, such as mesoderm formation during gastrulation and growth. In vertebrates, the Fgf superfamily consists of 22 family members, whereas only few FGFs are contained in the less complex genomes of insects and worms. In the recently sequenced genome of the beetle Tribolium, we identified four Fgf family members representing three subfamilies. Tribolium has Fgf1 genes that are absent in Drosophila but known from vertebrates. By phylogenetic analysis and microsynteny to Drosophila, we further classify Tc-fgf 8 as an ancestor of pyramus and thisbe, the fly Fgf8 genes. Tc-fgf8 expression in the growth zone suggests an involvement in mesoderm formation. In the embryonic head, expression of Tc-fgf8 subdivides the brain into a larger anterior and a smaller posterior region. The Fgf Tc-branchless is expressed in the embryonic tracheal placodes and in various gland-like structures. The expression patterns of the only Tribolium Fgf receptor and the adaptor molecule Downstream-of-Fgfr are largely congruent with Tc-Fgf8 and Tc-bnl. Thus, in contrast to Drosophila, only one Fgf receptor canalises Fgf signalling in different tissues in Tribolium. Our findings significantly advance our understanding of the evolution of Fgf signalling in insects.     

JAK-STAT signalling is required throughout telotrophic oogenesis and short-germ embryogenesis of the beetle Tribolium

In Drosophila, the JAK-STAT signalling pathway regulates a broad array of developmental functions including segmentation and oogenesis. Here we analysed the functions of Tribolium JAK-STAT signalling factors and of Suppressor Of Cytokine Signalling (SOCS) orthologues, which are known to function as negative regulators of JAK-STAT signalling, during telotrophic oogenesis and short-germ embryogenesis. The beetle Tribolium features telotrophic ovaries, which differ fundamentally from the polytrophic ovary of Drosophila. While we found the requirement for JAK-STAT signalling in specifying the interfollicular stalk to be principally conserved, we demonstrate that these genes also have early and presumably telotrophic specific functions. Moreover, we show that the SOCS genes crucially contribute to telotrophic Tribolium oogenesis, as their inactivation by RNAi results in compound follicles. During short-germ embryogenesis, JAK-STAT signalling is required in the maintenance of segment primordia, indicating that this signalling cascade acts in the framework of the segment-polarity network. In addition, we demonstrate that JAK-STAT signalling crucially contributes to early anterior patterning. We posit that this signalling cascade is involved in achieving accurate levels of expression of individual pair-rule and gap gene domains in early embryonic patterning.     

Molecular mechanisms of determination in Drosophila

A number of Drosophila proteins have been identified that play key roles in the establishment of active or inactive states of selector gene expression. Interactions between these proteins and their target selector genes are beginning to be understood, shaping our molecular view as to how stable determination of cells is achieved.     

同源盒基因(Hox)与哺乳动物生殖

哺乳动物的同源盒基因（Hox）与果蝇的同源异形基因是同源基因,该基因编码的DNA片段含183碱基对,转录由61个氨基酸残基组成的蛋白质保守结构域,称同源异型域.Hox基因碱基顺序及在染色体中的位置都是高度保守的.Hox基因在体节结构分化等空间信息调控中起着重要作用,按特异的空间模式赋予每一体节其自身的特点.近年来的研究表明,Hox基因不但影响胚胎发育,而且与成体生殖系统分化有关,在着床期子宫接受态的建立及子宫蜕膜反应的发生等生殖过程中起着重要的调节作用.     

Genome-wide acceleration of protein evolution in flies (Diptera)

Background

The rate of molecular evolution varies widely between proteins, both within and among lineages. To what extent is this variation influenced by genome-wide, lineage-specific effects? To answer this question, we assess the rate variation between insect lineages for a large number of orthologous genes.

Results

When compared to the beetle Tribolium castaneum, we find that the stem lineage of flies and mosquitoes (Diptera) has experienced on average a 3-fold increase in the rate of evolution. Pairwise gene comparisons between Drosophila and Tribolium show a high correlation between evolutionary rates of orthologous proteins.

Conclusion

Gene specific divergence rates remain roughly constant over long evolutionary times, modulated by genome-wide, lineage-specific effects. Among the insects analysed so far, it appears that the Tribolium genes show the lowest rates of divergence. This has the practical consequence that homology searches for human genes yield significantly better matches in Tribolium than in Drosophila. We therefore suggest that Tribolium is better suited for comparisons between phyla than the widely employed dipterans.     

Expression of <Emphasis Type="Italic">Pax group III</Emphasis> genes in the honeybee (<Emphasis Type="Italic">Apis mellifera</Emphasis>)

Pax group III genes are involved in a number of processes during insect segmentation. In Drosophila melanogaster, three genes, paired, gooseberry and gooseberry-neuro, regulate segmental patterning of the epidermis and nervous system. Paired acts as a pair-rule gene and gooseberry as a segment polarity gene. Studies of Pax group III genes in other insects have indicated that their expression is a good marker for understanding the underlying molecular mechanisms of segmentation. We have cloned three Pax group III genes from the honeybee (Apis mellifera) and examined their relationships to other insect Pax group III genes and their expression patterns during honeybee segmentation. The expression pattern of the honeybee homologue of paired is similar to that of paired in Drosophila, but its expression is modulated by anterior–posterior temporal patterning similar to the expression of Pax group III proteins in Tribolium. The expression of the other two Pax group III genes in the honeybee indicates that they also act in segmentation and nervous system development, as do these genes in other insects.     

White gene expression,repressive chromatin domains and homeotic gene regulation in Drosophila

The use of Drosophila chromosomal rearrangements and transposon constructs involving the white gene reveals the existence of repressive chromatin domains that can spread over considerable genomic distances. One such type of domain is found in heterochromatin and is responsible for classical position-effect variegation. Another type of repressive domain is established, beginning at specific sequences, by complexes of Polycomb Group proteins. Such complexes, which normally regulate the expression of many genes, including the homeotic loci, are responsible for silencing, white gene variegation, pairing-dependent effects and insertional targeting.